Non-lytic baculovirus

ABSTRACT

A baculovirus that infects a host cell without lyzing the host cell and related protein expression method. Within the scope of this invention are in vitro and in vivo methods for detecting protein folding or a cell lysis activity of a sample. Also within the scope of this invention is a method of screening for a compound for treating a disease associated with misfolding of a protein.

RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional ApplicationSer. No. 60/445,951, filed on Feb. 7, 2003, which is incorporated byreference in its entirety.

BACKGROUND

[0002] Baculoviruses are a group of rod-shaped, enveloped,double-stranded DNA viruses having a circular, supercoiled genomevarying from 90 to 160 kb in size. They have been successfully used forefficient expression of engineered proteins. A baculovirus system ismore attractive than other protein expression systems because of itshigh level expression, posttranslational modification ability, andsafety for use in humans (Smith et al., 1983, Mol. Cell Biol. 3,2156-2165).

[0003] However, the conventional baculovirus expression system has onedisadvantage. More specifically, host cells infected with baculoviruseslyse upon maturation of viral progenies. During the cell lysis, (1) theengineered protein leaks and is difficult to recover, leading to lowprotein yields; (2) the host cell lysosomes break down and releaseproteases, causing degradation of the engineered protein; (3)endoplasmic reticules and Golgi bodies also break down, resulting inpoor posttranslational modification and blockade of intracellulartrafficking of a membrane or secretory engineered protein. Thus, thereis a need for a non-lytic baculovirus.

SUMMARY

[0004] This invention is based on an unexpected discovery of a number ofnon-lytic baculovirus mutants.

[0005] Accordingly, one aspect of this invention features a baculovirusthat infects host cells, e.g., insect cells, without lyzing them. Thatis, the majority (i.e., at least 50%) of the host cells infected withthe baculovirus are not lyzed upon maturation of viral progenies. In oneembodiment, the baculovirus contains an exogenous nucleic acid sequencethat encodes a polypeptide. An exogenous nucleic acid sequence refers toany nucleic acid sequence that is not part of the baculovirus genome innature. One can infect a host cell with such a baculovirus to expressthe polypeptide. This polypeptide can be a fusion protein that containsa fluorophore. A fluorophore refers to a single fluorescent amino acidresidue (e.g., tryptophan, lysine, cystine, or their fluorescentderivatives) or a fluorescence peptide sequences having at least 2(e.g., 3, 4, 5, 10, 50, and 100) amino acid residues, i.e., fluorophoredomain. Examples of a fluorophore domain include enhanced yellowfluorescence protein (EYFP), enhanced cyan fluorescence protein (ECFP),enhanced green fluorescence protein (EGFP), and DsRed. Thisfluorophore-containing fusion protein can be used to detect proteinfolding in vitro and in vivo as described below.

[0006] Another aspect of the invention features a method for detectingprotein folding. The method requires the use of a protein that containsa donor fluorophore domain and an acceptor fluorophore domain, e.g.,ECFP and EYFP. The two fluorophore domains are disposed so that, whenthe protein is folded, they are in close proximity to allow resonanceenergy transfer between them. The method includes monitoringfluorescence emission change of the acceptor fluorophore domain uponirradiation of the donor fluorophore domain with an excitation light.The change is a function of the protein folding.

[0007] The invention also features a method for detecting proteinfolding in a cell. This method is identical to the method justdescribed, except that it requires the use of a cell expressing aprotein that contains a donor fluorophore and an acceptor fluorophore.In one embodiment, these fluorophores are two different fluorescenceprotein domains, e.g., ECFP and EYFP. The cell is a bacterial, a yeast,an insect, a plant, or a mammalian cell. Preferably, it is an insectcell.

[0008] The invention further features a method of screening for acompound for treating a disease associated with misfolding of a protein,such as cystic fibrosis, a disorder associated with misfolded cysticfibrosis transmembrane conductance regulator. The method requiresincubating in a first medium a compound and a plurality of cells thathave a protein linked to a donor fluorophore and an acceptorfluorophore, such as two different fluorescence protein domains (e.g.,ECFP and EYFP). The two fluorophores are disposed so that, when theprotein is folded, they are in close proximity to allow resonance energytransfer between them. The cells are bacterial, yeast, insect, plant, ormammalian cells, or, preferably, insect cells.

[0009] The just-described method includes determining the efficacy ofthe compound for treating the disease by monitoring cells emittingfluorescence from the donor or acceptor fluorophore upon irradiation ofthe donor fluorophore with an excitation light. In one embodiment, thedetermining step is conducted by identifying a percentage of cellsemitting the fluorescence of the acceptor fluorophore. The compound isdetermined to be effective in treating the disease if the percentage ofcells emitting fluorescence from the acceptor fluorophore is higher thanthat determined in the same manner on cells in a second medium, exceptthat the second medium does not contain the compound. In anotherembodiment, the determining step is conducted by identifying apercentage of cells emitting the fluorescence of the donor fluorophore.The compound is determined to be effective in treating the disease ifthe percentage of cells emitting fluorescence from the donor fluorophoreis lower than that determined in the same manner on cells in a secondmedium, except that the second medium does not contain the compound. Inyet another embodiment, the determining step is conducted by identifyinga resonance energy transfer efficiency of the cells. The compound isdetermined to be effective in treating the disease if the resonanceenergy transfer efficiency is higher than that determined in the samemanner on cells in a second medium, except that the second medium doesnot contain the compound. The resonance energy transfer efficiency isderived using the following formula:${{Resonance}\quad {energy}\quad {transfer}\quad {efficiency}} = {1 - \frac{I_{b}}{I_{a}}}$

[0010] One can obtain the intensities of the donor fluorophore inpresence and absence of the energy transfer (Ia and Ib), respectively,according to the method described in the Example below or other methodsknown in the art.

[0011] In a further aspect, the invention features a method of detectinga cell-lysis activity of a sample. The method includes (1) incubating ina first medium a sample and a plurality of cells that have a proteincontaining a fluorophore, e.g., ECFP, EGFP, EYFP, or DsRed; and (2)determining a percentage of cells emitting fluorescence upon irradiationof the fluorophore with an excitation light. The sample is determined tohave a cell-lysis activity if the percentage of cells emittingfluorescence is lower than that determined in the same manner on cellsin a second medium, except that the second medium does not contain thesample.

[0012] The invention features another method for detecting a cell-lysisactivity of a sample. The method requires the use of a plurality ofcells that have a protein containing a donor fluorophore and an acceptorfluorophore, such as two different fluorescence protein domains (e.g.,ECFP and EYFP). The two fluorophores are disposed so that, when theprotein is folded, they are in close proximity to allow resonance energytransfer between them. The method includes (1) incubating in a firstmedium a sample and the cells; (2) determining the cell-lysis activityof the sample by monitoring cells emitting fluorescence from the donoror acceptor fluorophore upon irradiation of the donor fluorophore withan excitation light. In one embodiment, the determining step isconducted by identifying a percentage of cells emitting the fluorescenceof the acceptor fluorophore. The sample is determined to have acell-lysis activity if the percentage of cells emitting fluorescencefrom the acceptor fluorophore is lower than that determined in the samemanner on cells in a second medium, except that the second medium doesnot contain the sample. In another embodiment, the determining step isconducted by identifying a percentage of cells emitting the fluorescenceof the donor fluorophore. The sample is determined to have a cell-lysisactivity if the percentage of cells emitting fluorescence from the donorfluorophore is higher than that determined in the same manner on cellsin a second medium, except that the second medium does not contain thesample. In yet another embodiment, the determining step is conducted byidentifying a resonance energy transfer efficiency of the cells. Thesample is determined to have a cell-lysis activity if the resonanceenergy transfer efficiency is lower than that determined in the samemanner on cells in a second medium, except that the second medium doesnot contain the sample.

[0013] In both cell-lysis detecting methods, the cells are bacterial,yeast, insect, plant, or mammalian cells, or preferably, insect cells.They can be used to screen for agents, e.g., compounds ormicroorganisms, that kill unwanted cells, such as infectious bacterialcells, yeast cells, fungus cells, or cancerous cells.

[0014] The details of one or more embodiments of the invention are setforth in the description below. Other features, objects, and advantagesof the invention will be apparent from the description and drawings, andfrom the claims.

DETAILED DESCRIPTION

[0015] The present invention relates to a non-lytic baculovirus. Hostcells infected with it do not lyse. An expression system based on thisnon-lytic baculovirus has several advantages over conventional systems.

[0016] First, the engineered protein expressed in the host cells doesnot leak out of the host cells. Second, the host cell lysosomes do notbreak down to release proteases and, as a result, one can harvest theengineered protein without using expensive protease inhibitors. Finally,as endoplasmic reticules and Golgi bodies in the host cells remainintact and efficiently modify and transport engineered protein, thissystem is useful for producing glycoprotein, membrane protein, orsecretory protein.

[0017] To produce an engineered protein, one can simply infect a hostcell with a baculovirus of this invention, which contains an exogenousnucleic acid sequence encoding the protein using standard techniques.See, e.g., Pfeifer et al., 1997, Gene 188:183-190; and Clem et al.,1994, J Virol 68:6759-6762. Suitable host cells may vary depending onthe designs of systems or consideration of specificity. Examples ofsuitable host cells include, but are not limited to, the cells derivedfrom species ranging from insects to vertebrates. Preferred host cellsare insect-derived cells and mammalian cells. Examples of theinsect-derived cells include S2 cells, Kc cells, and C6/36 cells.Suitable mammalian cells include primary cells or cell lines frommurine, rat, rabbit, porcine, or human sources. The expressed proteincan be therapeutic protein. They can be used to express in a subject(e.g., a mammal, such as a human) a nucleic acid sequence encoding aprotein that corrects a deficiency in gene expression. Examples of theproteins include vaccines, antibodies, biologically active peptides,cytokine and their receptors, growth factors and their receptors, andenzymes.

[0018] The baculoviruses of this invention can be used in detecting thefolding of a protein of interest. Conventional analysis of proteinfolding is based on in vitro spectroscopic and thermodynamic methods(Garcia-Mira et. al., Science 298, 2191-2195). These in vitro methodsrequire laborious protein purification, during which the proteins tendto be unfolded or degraded. An in vivo system is expected to allow oneto better study protein folding. The baculoviruses of this inventionprovide such an in vivo system. More specifically, one can (1) design afusion protein containing the protein of interest and a pair of donorand acceptor fluorophores, (2) express the fusion protein in a hostcell, and (3) study the folding of the protein of interest by examiningthe fluorescence resonance energy transfer (FRET) between the twofluorophores.

[0019] The efficiency of FRET can be derived from the formula descriedabove. It is determined by the angular orientation and the distancebetween the donor and acceptor fluorophores. Typically, FRET takesplaces over a distance less than 100 Å, which is comparable to thedimension of most folded proteins. Thus, it allows one to study thefolding status of a protein. Take the ECFP/EYFP pair for example, theirFörster distance R0 (a distance at which the FRET efficiency is 50%) is49 Å. FRET between them remains detectable over a distance of 78 Å(Bastiaens and Jovin 1996, Proc Natl. Acad. Sci. 93, 8407-8412;Bastiaens and Harpur 2001, Molecular Cloning—A laboratory manual, 3rd.Ed. Sambrook, J. and Russell, D. W. ed., A 18.69).

[0020] To detect the folding status of a protein, a donor fluorophoreand an acceptor fluorophore are fused to or inserted into the protein sothat (1) when the protein is unfolded/denatured, the two fluorophoresare too far away to allow FRET, and (2) when it is folded and becomesmore compact, the two fluorophores are brought closer enough for FRET totake place. The FRET can be detected by measuring the fluorescenceemission of either the acceptor or the donor fluorophore according tothe method described in the actual examples below or by other techniquesknown in the art. When a protein's N- and C-termini are in the samedirection, the two fluorophores can simply be fused to the two termini,respectively. Indeed, many proteins have N- and C-termini in the samedirection (see, e.g., Zhao, et al., 2001, J. Biol. Chem. 276,12120-12127; Riedl, et al., 2001, Proc. Natl. Aced. Sci. USA 98,14790-14795; and Cierpicki and Otlewski, 2002, J. Mol. Biol. 321,647-658.). Otherwise, one can determine the positions to insert the twofluorophores based on the 3-D structural data of a protein, which can beobtained using techniques well known in the art, such as NMR and X-raycrystallography. See, e.g., Conti et al., 1996, Structure 4, 287-298.

[0021] The just-described detecting method can be used in real time tomonitor the folding-denature status of a protein in response to variousgenetic changes (e.g., point mutation and presence of chaperone protein)or environmental changes (e.g., pH, temperature, and presence of certaincompounds). Accordingly, it can be used to screening for conditions orcompounds that enhance the folding of a protein of interest.

[0022] It is known that some genetic mutations compromise normal proteinfolding and lead to unfolded or misfolded protein. These proteinscontribute to various disorders, ranging from cystic fibrosis toneurodegenerative diseases. See, e.g., Massiah et al., 1999,Biochemistry 38, 7453-7461; Travers, et al., 2000, Cell 101, 249-258;and Sherman and Goldburg, 2001, Neuron 29:15-32. Thus, compounds thatfacilitate protein folding can be used in treating such diseases.

[0023] To identify such compounds, one can use the above-describedfusion protein that contains a donor fluorophore and acceptorfluorophore or cells (e.g., bacterial, yeast, insect, plant, ormammalian cells) containing such fusion protein. In one example, thecells are incubated in a first medium containing a test compound and asecond medium free of the compound, respectively. One then (1) monitorsin both media the cells emitting fluorescence from the donor fluorophoreupon irradiation of the donor fluorophore with an excitation light and(2) identifies a percentage of cells emitting the fluorescence of thedonor fluorophore. The compound is determined to be effective intreating a disease associated with misfolded protein if the percentageof cells emitting fluorescence from the donor fluorophore in the firstmedium is lower than that determined in the same manner on the cells inthe second medium. Alternatively, one can identify a percentage of cellsemitting the fluorescence of the acceptor fluorophore or the FRETefficiency on the cells in both media. The compound is determined to beeffective in treating the disease if the percentage or FRET efficiencydetermined on the cells in the first medium is higher than thatdetermined in the same manner on the cells in the second medium.

[0024] Compounds to be screened can be obtained using any of thenumerous approaches in combinatorial library methods known in the art.Such libraries include: peptide libraries, peptoid libraries (librariesof molecules having the functionalities of peptides, but with a novel,non-peptide backbone that is resistant to enzymatic degradation),spatially addressable parallel solid phase or solution phase libraries,synthetic libraries obtained by deconvolution or affinity chromatographyselection, the “one-bead one-compound” libraries, and antibodylibraries. See, e.g., Zuckermann et al. (1994) J. Med. Chem. 37,2678-85; Lam (1997) Anticancer Drug Des. 12, 145; Lam et al. (1991)Nature 354, 82; Houghten et al. (1991) Nature 354, 84; and Songyang etal. (1993) Cell 72, 767. Examples of methods for the synthesis ofmolecular libraries can be found in the art, for example, in: DeWitt etal. (1993) Proc. Natl. Acad. Sci. USA 90, 6909; Erb et al. (1994) Proc.Natl. Acad. Sci. USA 91, 11422; Zuckermann et al. (1994) J. Med. Chem.37, 2678; Cho et al. (1993) Science 261, 1303; Carrell et al. (1994)Angew. Chem. Int. Ed. Engl. 33, 2059; Carell et al. (1994) Angew. Chem.Int. Ed. Engl. 33, 2061; and Gallop et al. (1994) J. Med. Chem. 37,1233.Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13, 412-421), or on beads (Lam (1991) Nature 354,82-84), chips (Fodor (1993) Nature 364, 555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. No. 5,223,409), plasmids (Cull et al.(1992) Proc. Natl. Acad. Sci. USA 89, 1865-1869), or phages (Scott andSmith (1990) Science 249, 386-390; Devlin (1990) Science 249, 404-406;Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382; Felici(1991) J. Mol. Biol. 222, 301-310; and U.S. Pat. No. 5,223,409).

[0025] This invention also features a method for detecting a cell-lysisactivity of a sample. This method can be used to screen for cell lysisagents. To practice this method, one needs cells expressing a proteincontaining at least one fluorophore. One can use bacterial, yeast,fungal, insect, plant, or mammalian cells. The agents thus identifiedcan be used to kill unwanted cells, such as infectious bacterial cells,yeast cells, fungus cells, or cancerous cells.

[0026] In one embodiment, cells expressing a protein containing onefluorophore are used. Two groups identical cells are incubated in afirst medium containing a test sample and in a second medium free of thesample, respectively. Upon irradiation of the fluorophore with anexcitation light, cells emitting fluorescence from the fluorophore aremonitored to determine the percentage of the cells emitting thefluorescence. The sample is determined to have a cell-lysis activity ifthe percentage determined on the cells in the first medium is lower thanthat determined in the same manner on the cells in the second medium.

[0027] In other embodiments, one uses cells expressing a fusion proteincontaining a donor fluorophore and an acceptor fluorophore. He canrespectively incubate the cells in a first medium containing a testsample and in a second medium free of the sample. Upon irradiation ofthe donor fluorophore with an excitation light, he monitors in bothmedia cells emitting fluorescence from the donor fluorophore to identifya percentage of cells emitting the fluorescence of the donorfluorophore. The sample is determined to have a cell-lysis activity ifthe percentage is higher than that determined in the same manner on thecells in the second medium. Alternatively, he can identify a percentageof cells emitting the fluorescence of the acceptor fluorophore or theFRET efficiency on the cells in both media. The sample is determined tohave a cell-lysis activity if the percentage or FRET efficiencydetermined on the cells in the first medium is lower than thatdetermined in the same manner on the cells in the second medium.

[0028] The examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications cited herein arehereby incorporated by reference in their entirety.

[0029] Cells and Viruses

[0030] The Spodoptera frugiperda IPLB-Sf21 (Sf21) cell line was culturedin a TNM-FH insect medium containing 8% heat-inactivated fetal bovineserum (Lee et al., 1998, J. Virol. 72, 9157-9165 and Lin et al., 1999,J. Virol. 73, 128-139). It was used for the propagation and infection ofwild type and recombinant baculoviruses. All viral stocks were preparedand titers were determined according to the standard protocol describedby O'Reilly et al., 1994, Baculovirus Expression Vectors: A LaboratoryManual, Oxford University Press, New York.

[0031] Construction of Plasmids and Viruses

[0032] Plasmid pAB_(h)cmEpL, which contains two promoters to drive theexpression of two different foreign proteins, was constructed asfollows. The coding sequence of EGFP, derived from pEGFP-1 (Clontech,Palo Alto, Calif.), was inserted into a pBacPAK8 transfer vector(Clontech). The EGFP coding sequence was under the control of a CMVminimal promote, which was enhanced by an hr 1 sequence (Lo et al.,2002, J. Biol. Chem. 277:5256-5264.). Another DNA sequence encodingfirefly luciferase (LUC) was inserted into the same transfer vector andwas under the control of polyhedrin promoter. The resultant plasmid,pAB_(h)cmEpL, was co-transfected with vAcRP23.Laz (PharMingen, SanDiago, Calif.), a linearized viral DNA of AcMNPV, into Sf21 cells usingLipofectin (Invitrogen, Carlsbad, USA) to produce recombinant virusvAB^(h)cmEpL.

[0033] To generate a concatenated fusion gene, eyfp-luc-ecfp, a DNAfragment encoding LUC was prepared using PCR from a pTRE-Luc vector(Clontech) with primer pairs: forward primer,5′-GAAGATCTTTGGTCCCTCGTGGAAGCATGGAAGACGCCAAAAACATA-3′ and reverseprimer, 5′-CACCGGTCCATGATGATGATGATGATGCAATTCCACTTTCCGCCCTT-3′. Theforward primer was designed to contain a Bgl II site (underlined) and athrombin cutting sequence (bold) just upstream of the initiation codon.The reverse primer had an Age I site (underlined) downstream of thehexa-histidine tag (italics). After PCR amplification, the products wereligated into a pCR-Blunt II-TOPO vector (Invitrogen), to generate anintermediate plasmid. This intermediate plasmid, pBTII-Luc, was digestedby Bgl II and Age I, and the released fragment was inserted into apECFP-N1 vector (Clontech), to generate a plasmid containing theluc-ecfp fusion sequence. The resultant plasmid, pcLC, was used as atemplate to amplify the luc-ecfp fusion gene by PCR with the forwardprimers just described and another reverse primer,5′-GGGGTACCAATATTAACGCTTAC-3′ (Kpn I site underlined). The PCR productswere inserted into a pCR-Blunt II-TOPO vector to prepare anotherintermediate plasmid containing luc-ecfp fusion gene, from which a BglII and Kpn I-digested fragment was cloned into the Bgl II-Kpn I site ofa pEYFP-C1 vector (Clontech). The resultant plasmid, pcYLC, containedthe eyfp-luc-ecfp tandem fusion gene that was driven by an hr 1sequence-enhanced minimal CMV promoter. The fusion gene encoded a fusionprotein EYFP-LUC-ECFP (YLC), in which LUC was sandwiched between aN-terminally tagged EYFP and a C-terminally tagged ECFP.

[0034] The just-described pcYLC was cut by Nhe I and Not I to release afragment containing the eyfp-luc-ecfp coding sequence. This fragment wasthen cloned into the Xba I-Not I site of a pBacPAK8 vector. Theresultant transfer plasmid, pABpYLC, contained the eyfp-luc-ecfp codingsequence under the control of the polyhedrin promoter. The polyhedrinpromoter of pABpYLC was then replaced with the above-described hrlsequence-enhanced minimal CMV promoter to make a pAB^(h)cmYLC transfervector.

[0035] Two additional transfer plasmids, pABpC and pABpY, were createdby cutting pECFP-N1 with Pst I and Not I and digesting pEYFP-C1 with NheI and Kpn I, and ligating the fragments into the Pst I/Not I and XbaI/Kpn I sites of pBacPAK8 vectors, respectively. pABpLC was prepared bythe removal of eyfp from pABpYLC with Bgl II and BamH I digestion. Itcontained the luc-ecfp fusion gene

[0036] Recombinant baculoviruses were prepared by co-transfection 0.1 μgof various transfer plasmids described above (pABpYLC, pAB^(h)cmYLC,pABpLC, pABpC, and pABpY, respectively) and 0.1 μg of linearized AcMNPVviral DNA (BaculoGold, Pharmagen, San Diago, Calif.) into cells usingLipofectin. The resultant recombinant baculoviruses were named asvABpYLC, vAB^(h)cmYLC, vABpLC, vABpC, and vABpY, respectively.

[0037] Mutagenesis and Isolation of Nonlytic Baculovirus Mutants

[0038] General and saturated mutagenesis of the genome of Autographacalifornica nucleopolyhedrovirus (AcMNPV) was conducted to generatemutants. The mutants were then used to infect host cells. Since hostcells infected with nonlytic and lytic baculovirus viruses aremorphologically indistinguishable before cell lysis, it is difficult toisolate nonlytic mutants at the early phrase after infection. On theother hand, after cell lysis can be appreciated by standard cytologytechniques, too many virus particles are already in the medium and aredifficult to isolate pure virus clones. GFP in a host cell freely leaksout before cell lysis can be appreciated, it therefore was used as anindicator for identifying and isolating nonlytic viruses.

[0039] Sf21 cells (2×10⁵) were infected with vABhcmEpL at a multiplicityof infection (moi) of 1 TCID50 per cell and incubated at 26 or 33° C. inthe presence of 5-bromodeoxyuridine (BrdU) at concentrations of 10, 30,and 40 μg/ml. The culture media were harvested at 5 days post-infection(dpi), and excess BrdU was removed by dialysis in a buffer (137 mM NaCl,29 mM KCl, 4.3 mM Na₂HPO₄·7H₂₀, 1.4 mM KH₂PO₄, pH 7.2, plus 0.5% w/vBSA). Mutant viruses were purified by end-point dilution for threerounds on 96-well plates, and the purified viruses were used to infectindividual wells of 96-well plates. 25 μl medium from each well wasremoved and used to infect Sf21 cells. The infected cells were incubatedat 26° C. At 5 and 8 dpi, the expression of EGFP was examined using afluorocytometer to identify viral isolates that caused high cellularEGFP levels.

[0040] 11,603 clones were isolated. It is known that the baculovirusgenome has less than 154 genes/open reading frames. These 11,603 clonesshould be more than enough to cover all the possible viral genes, themutations of which would result in nonlytic infection. Among them, 118mutant clones showed reduction in cell lyses as indicated by theretention of EGFP in cells. In these 118 clones, 20 clones, i.e., A1,A2, A3, C4, 1028, 1044, 1053, 1071, 1081, 1085, 1091, 1094, 3058, 3074,PN8, PN9, PN19, PN23, PN24, and PN121, were further analyzed.

[0041] These viruses were different from the p35 mutant disclosed in Leeet al., 1998, Journal of Virology 72, 9157-9165, as all had intact p35gene. Further, after infection by a p35 mutant, most host cells (about95-99%) were lyzed. In contrast, after infection with one of the 21mutants, the majority of host cells remain intact. The cell lysis causedby these mutants was significantly less than that by the vABhcmEpLvirus. The results were even more significant if higher moi was used.Also, it was found that significant more EGFP proteins were produced byall these mutants than by vABhcmEpL. As CMVm promoter drove theexpression of EGFP, the results suggest that CMVm promoter is moreactive in cells infected with the mutants.

[0042] Each of the viruses described above had a LUC-encoding sequenceunder the control of the polyhedrin promoter, a very late promoter. Tostudy the activity of this promoter, the activity of luciferase wasexamined. More specifically, cells infected with each virus were lysedfor 25 min in 100 μl of a cell lysis reagent containing 100 mM potassium(pH 7.8), 1 mM EDTA, 10% glycerol, 1% Triton X-10, and 7 mMβ-mercaptoethanol. After centrifugation at 14,000 rpm for 10 min, thesupernatant or lysate (50 μl) was collected and mixed with 180 μl of LUCassay reagent containing 25 mM Tricine (pH 7.8), 15 mM potassiumphosphate (pH 7.8), 15 mM MgSO₄, 4 mM EGTA, 1 mM ATP, and 0.1 mMdithiothretitol. Fifty microliters of a luciferin (Promega, Madison,Wis.) solution was added into with the mixture, and relative light unitswere measured using a luminometer (Berthold, Lumat LB 9501, Bad Wildbad,Germany). The total protein concentration of the cell lysate wasdetermined using a Coomassie protein assay reagent (Pierce, Lselin,USA). Higher activities of the polyhedrin promoter were found in hostcells infected with clones 1081, 3058, PN8, PN9, PN19, PN23, PN24 andPN121.

[0043] Among the above-described 21 mutants, clone C4 leaded to theleast cell lysis and PN24 had the strongest polyhedrin promoteractivity. Both were further examined.

[0044] To generate nonlytic C4 recombinant viruses encoding anengineered protein YLC, pABpYLC and pAB^(h)cmYLC were transfected intoSf21 cells. The cells were then infected with the C4 nonlyticbaculovirus to generate recombinant viruses vC4pYLC and vC4^(h)cmYLC. Inthese baculoviruses, the sequences encoding YLC were under the controlsof polyhedrin promoter and a synthetic early promoter, respectively. Allrecombinant viruses were purified by three rounds of end-point dilution.

[0045] These recombinant nonlytic C4 baculoviruses and the lyticparental vABhcmEpL virus were used to infect Sf21 cells at 1 moi,respectively. At 5 dpi., the cells were examined by fluorescencemicroscopy. It was found that less than 40% of cells infected withvABhcmEpL contained EGFP protein. In contrast, more than 95% of cellsinfected with C4 baculoviruses contained EGFP. The result againindicates that the EGFP could be used to detect cell lysis before celllysis can be appreciated by standard microscopic techniques. It alsoindicates that conventional baculoviruses lead to leak of engineeredprotein upon cell lysis and lower protein yields.

[0046] Cells infected with the lytic and nonlytic baculoviruses werefurther examined by electron microscopy. It was found that nonlytic C4virus-infected cells had intact cytoplasmic membrane and ER. Incontrast, those infected with the lytic parental virus vABhcmEpL hadsevere damages in these organelles. ER is an important organelle forprotein modification, folding, sorting, and trafficking (Stevens andArgon, 1999, Seminars in Cell and Developmental Biology 10, 443-454.).Damaged ER leads to poor quality of engineered protein. The aboveresults indicate that nonlytic C4 baculovirus-infected cells producebetter folded engineered protein than those infected with lyticbaculovirus. In addition, folding of engineered protein requirescellular chaperone proteins. As these proteins may leak out fromlytically-infected cells but not from non-lytically infected ones, theengineered protein is better folded in the nonlytic baculovirus-hostcell system.

[0047] In vitro Protein Folding

[0048] FRET technology was used to examine the folding of luciferase(LUC), which contains 521 residues and has a molecular weight of 62 kD.In a folded LUC, the distance between its N- and C-terminus is about 40Å (Conti et al., 1996, Structure 4, 287-298). If its N and C-termini arefused to EYFP and ECFP, respectively, the distance between the centersof the two fluorescent proteins was estimated to be 40-60 Å. Assumingthat the mutual orientation of the two fluorophores was random andfreely tumbling, only a limited FRET. Hence, only tightly foldedluciferase could allow FRET between the two fluorophores.

[0049] The above-described YLC-encoding vectors were used to generate 4recombinant viruses: 2 conventional lytic vABpYLC and vAB^(h)cmYLC, and2 nonlytic vC4pYLC and vC4^(h)cmYLC. YLC fusion protein was generated inhost cells infected with these baculoviruses using standards techniquesand purified using a HisBind Kit (Novagen, Wisconcin, USA) and SephacrylS-200 (Pharmacia, Uppsala, Sweden) gel filtration resins. Its purity waschecked by SDS-PAGE, and the concentration was determined using a MicroBCA Kit (Pierce).

[0050] The fluorescence emission spectra of the YLC pro BOWMAN Series 2,Spectronics Unicam, Rochester, USA). The excitation wavelength was setat 436 nm, and the emission spectrum from 450 to 600 nm was recorded.For comparison, the fluorescence emission of the LUC-ECFP fusion protein(LC) and EYFP were measured after these proteins were excited by lightsof 436 and 510 nm, respectively. All spectra data were corrected forbackground fluorescence from the buffers. The emission spectrum showed apeak at 475 nm (cyan emission from ECFP) and, more importantly, anobvious emission peaked at 530 nm (yellow emission from EYFP). If LUC isproperly folded, ECFP (the donor) and EYFP (the acceptor) are in closeproximity to allow FRET between them, producing yellow fluorescenceemission. Otherwise, no yellow emission is detected. These resultsindicate that FRET took place in the fusion protein, which wasunexpected given the size of LUC (521 residues), the estimated distancebetween the centers of the two fluorescent proteins (40-60 Å), and theR0 value for ECFP/EYFP pair (49 Å).

[0051] To test the specificity of the FRET, the YLC fusion protein wasengineered to contain a thrombin digestion site between EYFP and therest of the fusion protein. After the EYFP domain was removed from therest of the YLC protein by a thrombin treatment, the yellow fluorescencewas abolished. This result indicates that the FRET is specific.

[0052] Further, the YLC fusion protein was incubated with urea (from 0 Mto 5 M) and monitored for its fluorescence. It was found that, as ureaconcentration increased, the intensity of the 475-nm fluorescenceemission increased, while that of the 530-nm emission decreased andreturned to the background level in the presence of 5 M urea. It isknown that fluorescent proteins, including ECFP and EYFP, are resistantto urea (Ward, 1998, Green Fluorescence Protein—Properties, application,and Protocols, Chalfie, M. and Kain, S. ed., pp45-75. New York:Wiley-Liss). In contrast, many other proteins, such as LUC, are not anddenature in presence of low concentration of urea (˜1 M) or guanidiniumchloride (Herbst et al., 1997, J. Biol. Chem. 272, 7099-7105). Thus,these results indicate that the change in the yellow emission from EYFPis a function of the folding/denaturing status of LUC.

[0053] In Vivo Protein Folding

[0054] The just-described nonlytic baculovirus-host cell system and FRETtechnology were used to examine folding of luciferase in Sf21 insectcells.

[0055] Sf21 cells were infected with various recombinant baculoviruses(vABpYLC, vABhcmYLC, vC4pYLC, and vC4hcmYLC) expressing engineeredfusion YLC proteins. They were then examined by laser scanning confocalfluorescence microscopy (Pascal LSM, Zeiss, Oberkochen, Germany), andpictures were taken using a ×40 objective len. The cells were thenexcited by a laser of 458 nm provided by an argon laser. Fluorescenceemission was separated as cyan channel (from 475 nm to 515 nm and yellowchannel (>530 nm). For discrimination, blue and yellow pseudocolor wasassigned to the cyan and yellow channels, respectively. For cellsemitting similar intensities of both cyan and yellow fluorescenceemissions, a combination of the two pseudocolors (pale-yellow or white)was assigned. For ECFP detection, fluorescence emission was collectedthrough a dual-wavelength beamsplitter at 458/514 nm and a longpassemission filter at 475 nm. For EYFP detection, the same beam splitterand a long pass emission filter at 530 nm were used. For simultaneousdetection of cyan and yellow fluorescences, the infected cells wereexcited by a laser of 458 nm. The acceptor yellow fluorescence wascollected using the 458/514-nm beam splitter and a long pass 530-nmemission filter, whereas the donor cyan fluorescence emission wasseparated by locating another 515-nm beam splitter and a band pass475-525 nm emission filter after the 458/514-nm beam splitter.

[0056] It was found that lytic baculovirus-infected cells showed eitherblue or yellow with some white emission. On the contrary, nonlyticbaculovirus-infected cells were mostly pale-yellow to white.

[0057] To determine the efficiency of the FRET of each baculovirus-hostcell system, photobleaching experiment was conducted. Sf21 cells wereinfected with vABpYLC, vAB^(h)cmYLC, vC4pYLC, and vC4^(h)cmYLC in thesame manner as described above. Selected infected cells were excited bya light of 458 nm. The intensities of cyan fluorescence (Ib) and yellowfluorescence emissions were measured. Afterwards, each cell wasphotobleached by an argon laser of 514 nm (excitation light for EYFP) tosaturate the EYFP. As a result, cyan fluorescence from the infected cellwas dequenched or recovered due to blockage of the FRET. Then, theintensity of cyan fluorescence (Ia) was measured. The FRET efficiencywas then calculated using the following formula:${{FRET}\quad {efficiency}} = {1 - {\frac{I_{b}}{I_{a}}.}}$

[0058] The efficiency reflects the fraction of cyan fluorescenceemission energy that is transferred to the yellow fluorescence protein.

[0059] It was found that the FRET efficiencies in vABpYLC-,vAB^(h)cmYLC-, vC4pYLC-, and vC4^(h)cmYLC-host cell systems were0.08±0.08, 0.12±0.1, 0.21±0.08, and 0.23±0.09, respectively. Thedifference between early and very late promoter lytic systems was lesssignificant (0.05<p<0.1). In contrast, the difference between the lyticand nonlytic systems was statistically significant (p<0.05). Theseresults indicate that YLC protein in the lytic baculovirus-host cellsystem is degraded. The loss of one or both of ECFP and EYFP domains, orthe linkage between them lead to a lower FRET efficiency. Accordingly,FRET efficiency, as well as the donor or acceptor fluorescence emission,reflects cell or protein lysis status, in addition to protein folding.The results also indicate that the nonlytic system described herein issuperior to the conventional system for studying protein folding.

[0060] Theoretically, cells emitting a cyan fluorescence have low FRETefficiencies. In contrast, those emitting pale-yellow or white cellsshould have satisfactory FRET efficiencies. In particular, cellsemitting yellow fluorescence should have the highest efficiencies, asall cyan fluorescence energy is transferred to EYFP. Some ABpYLC orvABhcmYLC-infected cells emitted yellow fluorescence. However, afterphotobleaching, they showed no fluorescence let alone enhanced cyanfluorescence, indicating no FRET. This discovery indicates that thatinfection of lytic vABpYLC or vABhcmYLC generated much of YLC fragments,instead of full length YLC. As a result, no FRET took place. Thus, theconvention baculovirus system is inferior to the non-lytic systemdescribed herein for FRET analysis.

[0061] It has been reported that the FRET between EGFP and EYFP has lowefficiencies of 0.10-0.15 (Harpur et al., Nature Biotechnol. 19,167-169). Given the R0 value of ECFP/EYFP pair (4.9 nm) and the size ofLUC, it was unexpected that the YLC fusion protein described above had ahigh FRET efficiency (>0.15). It is possible that this high FRETefficiency results from a favorable orientation of fluorophoretransition dipoles due to the proper and flexible linker molecule orfrom a large amount of EYFP due to the high protein yield of thebaculovirus expression system.

[0062] Alternatively, the higher efficiency may be an overestimation ofthe dimerization of fluorescence proteins (Zacharias et al., 2002,Science 296, 913-916.). Such dimerization or overcrowding might lead tointer-molecule energy transfer and in turn a high FRET efficiency. Toexamine whether this is the case, Sf21 cells were co-infected with tworecombinant baculoviruses bearing ecfp and eyfp genes at an moi of 10(to ensure coinfection of the same cells), and examined in the samemanner described above. It was found that only limited FRET efficiency(3%-8%) was achieved, suggesting that inter-molecule energy transferdoes not lead to high FRET efficiencies. Further, after adjusting forthis basal efficiency, the above described FRET efficiencies ofvABpYLC-, vABhcmYLC-, vC4pYLC-, and vC4hcmYLC-host cell systems (i.e.,0.08, 0.12, 0.21, and 0.23), were 0.00-0.05, 0.04-0.09, 0.13-0.18, and0.15-0.20, respectively. The adjusted efficiencies for the lytic andnonlytic system were even more statistically significant.

[0063] Proteins Expression

[0064] After infecting with wild type baculoviruses, host cells areeventually lysed at the very late phase of viral infection, a time closeto activation of the polyhedrin promoter and the expression of a largeamount of engineered protein. It is known that cellular machinery suchas foldase and chaperones is necessary for the proper folding ofengineered proteins. Without proper assistance with folding, they tendto aggregate (Herbst et al., 1997, J. Biol. Chem. 272, 7099-7105.). Itis also known that unfolded proteins are usually subject to degradation(Sherman and Goldburg, 2001, Neuron 29:15-32.). This may cause the lowFRET efficiencies in lytically infected cells described above. To testthis hypothesis, western blot analysis was conducted to evaluate the YLCproteins produced from the above-described four expression systems.

[0065] Protein samples were separated on a 10% SDS-PAGE, and thenelectrotransferred onto a PVDF membrane. The membrane was blocked inTris-buffered saline (TBS) containing 5% non-fat milk at roomtemperature for 4 h while being gently shaken on an orbital shaker. Themembrane was then incubated with a first antibody specifically againstLUC (1:2500, Sigma, St. Louis, Mo.) or GFP variants (1:1000; Clontech)in TBS containing 5% nonfat milk and 0.05% Tween-20 overnight at roomtemperature with constant shaking. Unbounded antibodies were removed bythree 10-min washes in TBS containing 0.1% Tween-20 (TTBS). The membranewas incubated with a secondary antibody, a 1:2500-diluted horseradishperoxidase-conjugated anti-rabbit IgG (Goat) (PerkinElmer, Wellesley,USA), for 1 h at room temperature. After four 15-min washes with TTBS,protein bands bounded by antibodies were visualized by chemicalluminescence (Western Lightning, PerkinElmer) and developed on Kodakfilm (Pharmacia).

[0066] It was shown that full-length YLC (ca. 110 kD) was only found incells infected with the nonlytic viruses vC4pYLC or vC4hcmYLC. Incontrast, serious protein degradation was found in cells infected withthe lytic viruses vABpYLC and vABhcmYLC. As to blotting using anti-GFPprotein, a major band at 27 kD was found in cells infected with vABpYLC,and well matched EYFP or ECFP made using the viruses vABpY and vABpC,indicating that the 110 kD YLC fusion protein was degraded. Theseresults indicate that engineered protein expressed from cells infectedwith the nonlytic baculovirus has better quality than that from cellsinfected with and conventional lytic baculovirus

[0067] Expression of Membrane Protein

[0068] An engineered membrane protein, CHL1-DsRed, was expressed usingthe above-described nonlytic baculoviruses, as well as wild typebaculoviruses. CHL1, an Arabidopsis thaliana membrane protein, is anitrate/chlorate transporter and a member of the proton-dependentoligopeptide transporter (POT) family. It has 12 transmembranealpha-helical and a cytosolic N-terminal domain (Tsai et al. 1993, Cell72(5): 705-13).

[0069] A DNA sequence encoding the first 131 amino acid residues of CHL1protein was synthesized according to the baculovirus genetic codes(Ranjan and Hasnain, 1995, Indian J. Biochem. Biophys. 32(6): 424-8;Levin and Whittome, 2000, J. Gen. Virol. 81(Pt 9): 2313-25). Thissequence, encoding 3 transmembrane alpha helixes, was engineered tocontain a Kozak consensus sequence to facilitate gene translation. Inaddition, its second peptide was mutated from serine (TCC) to glycine(GGC), and 8 nucleotides CCTCCACC was added 5′ end to the ATG codon.This DNA sequence was then cloned into a pDsRed vector (Clontech) andin-frame linked to the DsRed gene, which encoded a red fluorescentprotein. The resultant chll-DsRed fusion gene was amplified by PCR witha BamHI site-containing 5′-primer and a Not I site-containing 3′-primer.The product was subcloned into a pBakPAK8 vector to generate the vectorpABpCR. The CHL1-DsRed fusion gene was also amplified by PCR with an NheI-site-containing 5′-primer and a Bcl I-site-containing 3′-primer, andsubcloned into the pABhcmEpL vector described above to generate thevector pAB^(h)cmCR. In both vector, the fusion gene was under thecontrol of the hr enhanced CMV minimal promoter. The DNA sequence of anhsp70 (heat shock) promoter was cloned to the Sac II site of pDsRed. Thefragment containing this hsp70 promoter and the DsRed gene was thensubcloned into EcoRV site of the pBacPAK8 vector to generate pABhRpX.

[0070] The hydropathy plot of the CHL1-DsRed protein was generated usingKyte & D oolittle's method (Kyte and Doolittle, 1982, J. Mol. Biol.157(1): 105-32) and the Expert Protein Analysis System (ExPASy)(Gasteiger et al., 2003, Nucleic. Acids Res. 31(13): 3784-8.). Threehydrophobic domains in the N-terminus region were distinguishable. TheC-terminal DsRed domain exhibited much lower hydrophobicity. It waspredicted that this fusion protein, once expressed in a host cell,should be located in the plasma membrane via its N-terminal CHL1 domain,and its C-terminal DsRed domain protrudes from the plasma membrane.

[0071] The just-described pABpCR and pABhcmCR were cotransfected withgenomic DNAs of vAB^(h)cmEpL, the above-described C4, and PN24 usingstandard techniques to generate vABpCR, vAB^(h)cmCR, C4-pCR,C4-^(h)cmCR, PN24-pCR, PN24-^(h)cmCR, respectively. Selected viralclones were then amplified, undergone genomic DNA extraction, andsubjected to electrophoresis after HindIII digestion to confirm thequality of extracted DNA. Titered viral stocks were stored in screwsealed vials at 4° C.

[0072] Sf21 cells were infected with the recombinant viruses justdescribed. Their fluorescence levels were measured on 1 to 8 dpi. Themeasuring was repeated 3 times each day and 3 duplicates were made ineach. Nine sets of data were obtained, calculated, and plotted. It wasfound that cells infected with vC4^(h)cmCR had the highest fluorescencelevel. Also, those infected with vC4 pCR had higher level than thoseinfected with vPN24 pCR, vAB^(h)cmCR, or vABpCR.

[0073] The 3D models reconstructed from the confocal microscopy of Sf21cells infected with the nonlytic baculovirus resembled hollow spherescovered by red clusters on their surfaces. In contrast, the cellsinfected with vABhRpX gave a 3D model resembling a solid ball.Cross-sections of these 3D models confirmed the differences betweencells expressing membrane DsRed proteins and cytosolic DsRed proteins.

[0074] These results suggest that membrane protein is better processedand transported in the cells infected with non-lytic baculoviruses thanin those with conventional lytic baculoviruses. This advantage may notdue to a strong promoter, but to the intact intracellular organelles,such as the ER, Golgi complex, and lysosomes in the cells infected withnon-lytic baculoviruses.

Other Embodiments

[0075] All of the features disclosed in this specification may becombined in any combination. Each feature disclosed in thisspecification may be replaced by an alternative feature serving thesame, equivalent, or similar purpose. Thus, unless expressly statedotherwise, each feature disclosed is only an example of a generic seriesof equivalent or similar features.

[0076] From the above description, one skilled in the art can easilyascertain the essential characteristics of the present invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. Thus, other embodiments are also within the scope of thefollowing claims.

1 3 1 47 DNA Artificial Sequence Primer 1 gaagatcttt ggtccctcgtggaagcatgg aagacgccaa aaacata 47 2 47 DNA Artificial Sequence Primer 2caccggtcca tgatgatgat gatgatgcaa ttccactttc cgccctt 47 3 23 DNAArtificial Sequence Primer 3 ggggtaccaa tattaacgct tac 23

What is claimed is:
 1. A baculovirus that infects host cells withoutlyzing the host cells.
 2. The baculovirus of claim 1, wherein the hostcells are insect cells.
 3. The baculovirus of claim 1, comprising anexogenous nucleic acid sequence nucleic acid sequence encoding apolypeptide.
 4. The baculovirus of claim 3, wherein the polypeptidecontains a fluorophore.
 5. The baculovirus of claim 4, wherein thefluorophore is ECFP, EYFP, EGFP, or DsRed.
 6. A method of expressing apolypeptide in a host cell, comprising infecting the host cell with abaculovirus of claim 1, wherein the baculovirus contains an exogenousnucleic acid sequence nucleic encoding the polypeptide.
 7. A method fordetecting protein folding, comprising: providing a protein that containsa donor fluorophore domain and an acceptor fluorophore domain, the twofluorophore domains being disposed so that, when the protein is folded,they are in close proximity to allow resonance energy transfertherebetween; and monitoring fluorescence emission change of theacceptor fluorophore domain upon irradiation of the donor fluorophoredomain with an excitation light, the change being a function of theprotein folding.
 8. The method of claim 7, wherein the donor fluorophoredomain is ECFP and the acceptor fluorophore domain is EYFP.
 9. A methodfor detecting protein folding in a cell, comprising: expressing in acell a protein that contains a donor fluorophore and an acceptorfluorophore, the two fluorophores being disposed that, when the proteinis folded, they are in close proximity to allow resonance energytransfer therebetween; and monitoring fluorescence emission change ofthe acceptor fluorophore upon irradiation of the donor fluorophore withan excitation light, the change being a function of the protein folding.10. The method of claim 9 wherein the donor fluorophore and the acceptorfluorophore are two different fluorescence protein domains.
 11. Themethod of claim 10, wherein the donor fluorophore is ECFP and theacceptor fluorophore is EYFP.
 12. The method of claim 10, wherein thecell is a bacterial, a yeast, an insect, a plant, or a mammalian cell.13. The method of claim 12, wherein the cell is an insect cell.
 14. Themethod of claim 9, wherein the cell is a bacterial, a yeast, an insect,a plant, or a mammalian cell.
 15. The method of claim 14, wherein thecell is an insect cell.
 16. A method of screening for a compound fortreating a disease associated with misfolding of a protein, the methodcomprising: incubating in a first medium a compound and a plurality ofcells that have a protein linked to a donor fluorophore and an acceptorfluorophore, the two fluorophores being disposed so that, when theprotein is folded, they are in close proximity to allow resonance energytransfer therebetween; and determining the efficacy of the compound fortreating the disease by monitoring cells emitting fluorescence from thedonor or acceptor fluorophore upon irradiation of the donor fluorophorewith an excitation light.
 17. The method of claim 16, wherein thedetermining step is conducted by identifying a percentage of cellsemitting the fluorescence of the acceptor fluorophore, wherein thecompound is determined to be effective in treating the disease if thepercentage of cells emitting fluorescence from the acceptor fluorophoreis higher than that determined in the same manner on cells in a secondmedium, except that the second medium does not contain the compound. 18.The method of claim 17, wherein the wherein the donor fluorophore andthe acceptor fluorophore are two different fluorescence protein domains.19. The method of claim 18, wherein the donor fluorophore is ECFP andthe acceptor fluorophore is EYFP.
 20. The method of claim 16, whereinthe determining step is conducted by identifying a percentage of cellsemitting the fluorescence of the donor fluorophore, wherein the compoundis determined to be effective in treating the disease if the percentageof cells emitting fluorescence from the donor fluorophore is lower thanthat determined in the same manner on cells in a second medium, exceptthat the second medium does not contain the compound.
 21. The method ofclaim 20, wherein the wherein the donor fluorophore and the acceptorfluorophore are two different fluorescence protein domains.
 22. Themethod of claim 21, wherein the donor fluorophore is ECFP and theacceptor fluorophore is EYFP.
 23. The method of claim 16, wherein thedetermining step is conducted by a resonance energy transfer efficiencyof the cells, wherein the compound is determined to be effective intreating the disease if the resonance energy transfer efficiency ishigher than that determined in the same manner on cells in a secondmedium, except that the second medium does not contain the compound. 24.The method of claim 23, wherein the wherein the donor fluorophore andthe acceptor fluorophore are two different fluorescence protein domains.25. The method of claim 24, wherein the donor fluorophore is ECFP andthe acceptor fluorophore is EYFP.
 26. The method of claim 16, whereinthe wherein the donor fluorophore and the acceptor fluorophore are twodifferent fluorescence protein domains.
 27. The method of claim 26,wherein the donor fluorophore is ECFP and the acceptor fluorophore isEYFP.
 28. The method of claim 16, wherein the cells are bacterial,yeast, insect, plant, or mammalian cells.
 29. The method of claim 28,wherein the cells are insect cells.
 30. A method of detecting acell-lysis activity of a sample, comprising: incubating in a firstmedium a sample and a plurality of cells that have a protein containinga fluorophore; and determining a percentage of cells emittingfluorescence upon irradiation of the fluorophore with an excitationlight, wherein the sample is determined to have a cell-lysis activity ifthe percentage of cells emitting fluorescence is lower than thatdetermined in the same manner on cells in a second medium, except thatthe second medium does not contain the sample.
 31. The method of claim30, wherein the fluorophore is ECFP, EGFP, EYFP, or DsRed.
 32. A methodfor detecting a cell-lysis activity of a sample, comprising: incubatingin a first medium a sample and a plurality of cells that have a proteincontaining a donor fluorophore and an acceptor fluorophore, the twofluorophores being disposed so that, when the protein is folded, theyare in close proximity to allow resonance energy transfer therebetween;and determining the cell-lysis activity of the sample by monitoringcells emitting fluorescence from the donor or acceptor fluorophore uponirradiation of the donor fluorophore with an excitation light.
 33. Themethod of claim 32, wherein the determining step is conducted byidentifying a percentage of cells emitting the fluorescence of theacceptor fluorophore, wherein the sample is determined to have acell-lysis activity if the percentage of cells emitting fluorescencefrom the acceptor fluorophore is lower than that determined in the samemanner on cells in a second medium, except that the second medium doesnot contain the sample.
 34. The method of claim 33, wherein the donorfluorophore and the acceptor fluorophore are two different fluorescenceprotein domains.
 35. The method of claim 34, wherein the donorfluorophore is ECFP and the acceptor fluorophore is EYFP.
 36. The methodof claim 32, wherein the determining step is conducted by identifying apercentage of cells emitting the fluorescence of the donor fluorophore,wherein the sample is determined to have a cell-lysis activity if thepercentage of cells emitting fluorescence from the donor fluorophore ishigher than that determined in the same manner on cells in a secondmedium, except that the second medium does not contain the sample. 37.The method of claim 36, wherein the donor fluorophore and the acceptorfluorophore are two different fluorescence protein domains.
 38. Themethod of claim 37, wherein the donor fluorophore is ECFP and theacceptor fluorophore is EYFP.
 39. The method of claim 32, wherein thedetermining step is conducted by identifying a resonance energy transferefficiency of the cells, wherein the sample is determined to have acell-lysis activity if the resonance energy transfer efficiency is lowerthan that determined in the same manner on cells in a second medium,except that the second medium does not contain the sample.
 40. Themethod of claim 39, wherein the donor fluorophore and the acceptorfluorophore are two different fluorescence protein domains.
 41. Themethod of claim 40, wherein the donor fluorophore is ECFP and theacceptor fluorophore is EYFP.
 42. The method of claim 32, wherein thedonor fluorophore and the acceptor fluorophore are two differentfluorescence protein domains.
 43. The method of claim 42, wherein thedonor fluorophore is ECFP and the acceptor fluorophore is EYFP.
 44. Themethod of claim 32, wherein the cells are bacterial, yeast, insect,plant, or mammalian cells.
 45. The method of claim 44, wherein the cellsare insect cells.